Efficient production of bioactive proteins and peptides is a primary function of the biomedical and industrial biochemical industry. Bioactive peptides and proteins are used as curative agents in a variety of diseases such as diabetes (insulin), viral infections and leukemia (interferon), diseases of the immune system (interleukins), and red blood cell deficiencies (erythropoietin) to name a few. Additionally, large quantities of proteins and peptides are needed for various industrial applications including, for example, the pulp and paper industries, textiles, food industries, personal care and cosmetics industries, sugar refining, wastewater treatment, production of alcoholic beverages and as catalysts for the generation of new pharmaceuticals.
With the discovery and implementation of combinatorial peptide screening technologies new applications for small peptides having specific binding affinities have been developed. These technologies include bacterial display (Kemp, D. J.; Proc. Natl. Acad. Sci. USA 78(7): 4520-4524 (1981); yeast display (Chien et al., Proc Natl Acad Sci USA 88(21): 9578-82 (1991)), combinatorial solid phase peptide synthesis (U.S. Pat. No. 5,449,754; U.S. Pat. No. 5,480,971; U.S. Pat. No. 5,585,275 and U.S. Pat. No. 5,639,603), phage display technology (U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,571,698; and U.S. Pat. No. 5,837,500), ribosome display (U.S. Pat. No. 5,643,768; U.S. Pat. No. 5,658,754; and U.S. Pat. No. 7,074,557), and mRNA display technology (PROFUSION™; U.S. Pat. No. 6,258,558; U.S. Pat. No. 6,518,018; U.S. Pat. No. 6,281,344; U.S. Pat. No. 6,214,553; U.S. Pat. No. 6,261,804; U.S. Pat. No. 6,207,446; U.S. Pat. No. 6,846,655; U.S. Pat. No. 6,312,927; U.S. Pat. No. 6,602,685; U.S. Pat. No. 6,416,950; U.S. Pat. No. 6,429,300; U.S. Pat. No. 7,078,197; and U.S. Pat. No. 6,436,665)]
In particular, in biomedical fields small peptides are regarded as linkers for the attachment of diagnostic and pharmaceutical agents to surfaces (see U.S. Pat. App. Pub. No. 2003/0185870 to Grinstaff et al., and U.S. Pat. No. 6,620,419 to Linter), as well as in the personal care industry for the attachment of benefit agents to body surfaces such as hair and skin (see commonly-owned U.S. Pat. No. 7,220,405 to Huang et al., and U.S. Pat. App. Pub. No. 2003/0152976 to Janssen et al.), and in the printing industry for the attachment of pigments to print media (see commonly-owned U.S. Pat. App. Pub. No. 2005/0054752).
Some commercially useful peptides may be synthetically generated or isolated from natural sources. However, these methods are often expensive, time consuming and characterized by limited production capacity. The preferred method of peptide production is through the fermentation of recombinant microorganisms engineered to express the protein or peptide of interest. Although preferable to synthesis or isolation, recombinant peptide production has a number of obstacles to be overcome in order to be cost-effective. For example, peptides and in particular short peptides produced in a cellular environment are susceptible to degradation by native proteases in the cell. Additionally, the purification of some peptides may be difficult depending on the nature of the protein or peptide of interest and may result in poor yields.
One means to mitigate the difficulties associated with recombinant peptide production is the use of chimeric genetic constructs encoding chimeric proteins. The chimeric proteins may comprise at least one portion of the desired protein product fused to at least one portion comprising a peptide tag, referred to herein as “fusion proteins”. The peptide tag may be used to assist protein folding, post expression purification and/or protein passage through the cell membrane and to protect the protein from the action of degradative enzymes,
In many cases it is useful to express a peptide in insoluble form, particularly when the peptide of interest (POI) is a small peptide that is typically soluble under normal physiological conditions and/or subject to endogenous proteolytic degradation within the host cell. Production of the peptide in an insoluble form both facilitates simple recovery and protects the peptide from undesirable proteolytic degradation. One means to produce the peptide of interest in an insoluble form is to recombinantly produce the peptide as part of an insoluble fusion peptide by including at least one peptide tag (referred to herein as a “solubility tag” or “inclusion body tag”) that induces inclusion body formation. The fusion protein may include at least one cleavable peptide linker so that the peptide of interest can be subsequently recovered from the fusion protein. The fusion protein may include a plurality of inclusion body tags, cleavable peptide linkers, and regions comprising the peptide of interest.
Recombinant microbial peptide production often requires the ability to efficiently label, detect/monitor, and/or screen/select cells producing the desired fusion peptide. This ability is useful during both the strain development phase (i.e., identity strains/mutants/growth conditions that improve peptide production) and commercial production phase (i.e. process monitoring). During strain development, it is particularly desirable to identify and select strains exhibiting improved performance using a technique that is sensitive, fast, easy, and non-toxic to the recombinant cell, i.e., permits selection and subsequent growth of the selected cells, and amenable to high-throughput processing or screening.
Various fluorescent labeling and detection techniques have been reported in the art to monitor and/or measure peptide production, although many of these techniques are not cost-effective or suitable for in vivo labeling and detection, especially when producing small peptides. Giepmans et al. (Science 312:217-224 (2006)) reviews the fluorescent “toolbox” for assessing protein production/location and function. Many of the labeling techniques require the use of a targeting molecule to achieve specific labeling, e.g. fusion of small organic dyes and/or quantum dots to antibodies. However, such immunological techniques often require fixation and/or permeabilization and are not amenable to in vivo labeling, especially when one wants to select and grow the cells exhibiting an improvement in peptide production.
Another peptide labeling approach is the incorporation of a detectable fluorescent marker as part of the fusion construct. For example, fluorescent proteins such as green fluorescent protein (GFP) and yellow fluorescent protein (YFP) are often used to detect and/or measure recombinant peptide production. However, fusion constructs comprising a large fluorescent protein are time consuming because they require a significant fluorescence development period) and may place an additional metabolic burden on the microbial host cell. Fusion of a large fluorescent protein to the peptide of interest adversely affects the production efficiency of the peptide of interest, especially when the peptide of interest is small relative to the fluorescent protein. It is desirable to use a detectable marker that is small, easily detectable (sensitive with low background noise), and suitable for in vivo labeling and detection applications. In particular, a simple and effective in vivo labeling system that can be used in combination with a fluorescence activated cell sorter (FACS) for detection and/or selection is desirable.
The LUMIO™ protein detection system (Invitrogen Life Technologies, Carlsbad, Calif.) is based on the incorporation of a small tetracysteine tag (TC) that covalently binds to a biarsenical labeling reagent (e.g. FlAsH-EDT2 [LUMIO™ green]; ReAsh-EDT2 [LUMIO™ red]); and CHoXAsh-EDT2 (U.S. Pat. No. 5,932,474; U.S. Pat. No. 6,054,271; U.S. Pat. No. 6,831,160; U.S. Pat. No. 6,008,378; U.S. Pat. No. 6,451,564; U.S. Pat. No. 6,686,458; U.S. Pat. No. 7,138,503; EP1032837, EP1684073, U.S. Pat. App. Pub. No. 20050176065 A1; and Griffin et al., Science 281:269-271 (1998)). Covalent binding of the labeling reagent to the tetracysteine tag generates a highly fluorescent complex. The LUMIO™ detection system has been extensively used to fluorescently label eukaryotic proteins in vivo, especially mammalian cells and mammalian cell lines (Ho and Starnbach, Infect. Immunity, 73(2):905-911 (2005); Adams et al., JACS, 124:6063-6076 (2002); Stroffekova and Proenza, Eur. J. Physiol., 442:859-866 (2001); Rice et al., Nat. Biotechnol., 19:321-326 (2001); and Int'l App. Pub. No. WO2007/023184A1.
Griffin et al. (Meth. Enzymol., 327:565-578 (2000)) reports that labeling of intact bacterial cells requires much higher concentrations of the biarsenical labeling reagent in the presence of β-mercaptoethanol (2-ME) for several hours (unpublished data), suggesting that the labeling reagent cannot easily penetrate into prokaryotic cells.
Ignatova and Gierash (PNAS, 101 (2):523-528 (2004)) reports in vivo labeling of E. coli cells using a tetracysteine tag/biarsenical labeling reagent system wherein the fluorescence spectra was measured using a fluorometer. However, the labeling process required lysozyme pretreatment to make the outer membrane permeable to the labeling reagent.
A simple and cost effective process for in vivo labeling and detecting TC-tagged proteins produced within prokaryotic cells that does not require the use of undesirable compounds, e.g. β-mercaptoethanol, and/or a permeabilizing pretreatment, e.g. lysozyme treatment, has not been reported. In general, the use of permeabilizing agents and/or reducing agents is undesirable as the treated cells may be non-viable and/or undergo an unpredictable stress response that may influence peptide production and/or the cell's growth characteristics. This is particularly important when the goal of the labeling process is to identify and select viable cells suitable for use in further experiments.
Furthermore, many commercial applications for small bioactive peptides often require purified product. Many of these small peptides are produced in a recombinant prokaryotic host cell in the form of insoluble fusion peptides. A labeling system that is both effective for monitoring fusion peptide production and enables separation from the peptide of interest during subsequent bulk processing is needed.
The problem to be solved is to provide a process of in vivo labeling fusion peptides recombinantly produced within a prokaryotic cell, which is fast, efficient, sensitive, and does not require the use of permeabilized cells. That is, the process does not include the need to contact the cells with an undesirable agent to increase permeability in order to achieve effective in vivo labeling. Furthermore, the process should include a fast and effective means for detecting and/or selecting viable, labeled cells characterized by improved peptide production, e.g. the use of a fluorescence activated cell sorter to collect live cells. In one aspect, the process should be capable of selecting and isolating live cells suitable for use in further experiments and/or selections. In another aspect, the process may include repeatable steps (growth-labeling-detection-selection) that may include at least one round of mutagenesis to facilitate host cell optimization (e.g. increased peptide production).